Method and apparatus for ion axial spatial distribution focusing

ABSTRACT

The present invention provides a mass spectrometer including an ion source for generating pre-cursor ions, ion fragmentation means for generating fragment ions from the pre-cursor ions, a reflectron for focusing the kinetic energy distribution of the ions, and an ion detector wherein the mass spectrometer also includes axial spatial distribution focusing means which in use acts on the ions after the ion fragmentation means and before the reflectron, the axial spatial distribution focusing means being operable to reduce the spatial distribution of the ions in the direction of the ion optical axis of the spectrometer. Suitably the axial spatial distribution focusing means comprising a cell with two electrodes  52, 54  which may be apertures or high transmission grids. A pulsed electrostatic field is generated by applying a high voltage pulse  60  to the first electrode  52  at the time when the pre-cursor ions of interest  56, 58  have just passed into the pulser  50 . The second electrode  54  is maintained at 0V during this time.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/129,879 filed on Jul. 25, 2008, which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The present invention is concerned with methods and apparatus for massspectrometers, especially TOF (“time-of-flight”) mass spectrometers. Inparticular, the present invention relates to methods and apparatus forion axial spatial distribution focusing.

TOF mass spectrometry is an analytical technique for measuring the massto charge ratio of ions by accelerating ions and measuring theirtime-of-flight to a detector.

Two known methods of TOF mass spectrometry are matrix-assisted laserdesorption/ionization TOF mass spectrometry (“MALDI TOF” massspectrometry) and tandem TOF mass spectrometry (“TOF-MS/MS” massspectrometry). Maldi TOF-MS and TOF-MS/MS are long established asmethods of identifying macro-molecular compounds in biological systemsfor example.

In Maldi TOF-MS a laser pulse is focussed to a small spot (“laser spot”)on a mixture of a sample of the biological material and alight-absorbing matrix on a sample plate so that a pulse of ions isproduced.

This ion pulse is analysed and detected with a time-of-flight massspectrometer, TOF-MS, so that the mass to charge ratio of the ions ismeasured.

In TOF-MS/MS mass spectrometry, ions undergo fragmentation before theyare analysed and detected. The ions may be fragmented by meta-stabledecay (post-source decay, PSD) or by collision induced dissociation(CID), for example. TOF-MS/MS is useful because it allows analysis ofboth precursor ions (non-fragmented ions) and product ions (fragmentedions). TOF-MS/MS mass spectrometry can be used in combination with MALDITOF mass spectrometry. In other words, a MALDI ion source can be used ina mass spectrometer in which ions undergo fragmentation before they aredetected.

In the ion source of the TOF mass spectrometer, at the time ofextraction of the ions there are different distributions of the ionsthat characterise their initial direction, position and energy. Forexample, the range of radial position (distance from the ion opticalaxis) is determined by the spot size, as illustrated in FIG. 1. Thus,after desorption from the sample plate 1, ions 2, 4 are spaced from theion optical axis 6 (the main axis of the spectrometer) by a distance R.In the case of a MALDI source the size of R is dictated by the diameterof the “laser spot”, being the area from which ions are generated fromthe sample by the laser beam.

Each point in the ion source can generate a distribution in the initialdirection or at an angle to the ion optical axis, as illustrated in FIG.2. Thus, an ion 10 may possess a velocity having a radial component thatis such as to cause them to travel away from the source at an angle θ tothe ion optical axis 6. This characterises the expansion of the ionplume 12 outwards from the centre of the spot 14.

The ions are also produced with a range of initial energy or speed, asillustrated in FIG. 3. Thus, ions 20, 22 within the ion plume havedifferent energy or velocity, such that, for example, the energy E₁ ofion 20 may be smaller than the energy E₂ of ion 22.

In the case of a Maldi sample the axial velocity distributioncorresponds to a distribution in what is commonly known as the Jetvelocity, typically around a few hundred ms⁻¹.

There is also a spatial distribution of ions in the axial directionnormal to the sample surface, as illustrated in FIG. 4. This can be dueto the different starting positions of ions because of sample topographyand/or thickness. It can also be due to different starting times forions coupled with the axial velocity. Thus, ions 30, 32 are separated inspace in the axial direction (being parallel to the ion optical axis 6)by a distance Z.

Each of the distributions affects the performance of the TOF-MS (andTOF-MS/MS) and the result is measured by the width of the peak for asingle mass to charge which in turn determines the mass resolution.

The size of the effects can be controlled by various means. For example,the radial spatial distribution is set by the size of the focussed laserspot and controlled by the collimation of the ion optical lenses in themass spectrometer. Similarly, the effect of the angular distribution isalso controlled by the lenses in the ion optics.

It is possible to compensate for either the axial spatial distributionor the velocity distribution with pulsed extraction by using the spatialdistribution of the ions in combination with the pulsed electrostaticfield to produce a space focus in the flight tube [Time-of-Flight MassSpectrometer with Improved Resolution’ W. C. Wiley and I. H. McLaren,Rev. Sci. Instrum., 26, 1150 (1955)]. The space focus is a point whereall the ions in the velocity distribution come together at the sametime. The space focus can be at the detector in the case of a lineartime of flight or it can be the front focus of the ion mirror in areflectron time of flight.

However, in using pulsed extraction from the ion source, it is wellknown that only one axial distribution can be focussed at a time. Eitherthe initial axial spatial distribution or the initial axial velocitydistribution can be brought to a space focus but not both distributionssimultaneously.

In the case of orthogonal extraction of ions from a beam such as inelectrospray TOF-MS, the axial spatial distribution is focussed bypulsed extraction whilst the axial velocity distribution for the time offlight is negligible being in the orthogonal direction.

In an ion source such as a MALDI or SIMS ion source, the ions aredesorbed from the surface of a sample deposited on a plate by usingpulsed extraction, which focuses the velocity distribution. This workson the basis that the size of the initial axial spatial distribution ismuch less than the spatial distribution of the ions produced by thevelocity distribution during the delay time before pulsed extraction.However, this will only be the case if the sample is very thin (a fewmicrons) and/or if the laser power is very close to the threshold forgenerating ions so that they originate only from the surface of thesample.

Thus, using an appropriate design of ion optics to collimate the ionbeam coupled with a pulsed extraction ion source where the depth ofdesorbed sample is very thin and the laser power is very close to thethreshold, it is possible to achieve very high mass resolutions forTOF-MS.

In TOF-MS the ions extracted from the ion source arrive at the detectorintact so that the mass to charge ratio of the molecules from the sampleis measured. If the ions are made to break up into smaller pieces orfragments, in the field free region, it is possible with a reflectronTOF-MS to measure the mass to charge ratio of the fragment ions and socarry out TOF-MS/MS. This technique, also known as tandem TOF or TOF/TOFallows the analysis of the structure of the molecules desorbed from thesample. So, for example, the amino-acid sequence of a peptide or proteinsample can be determined from the TOF-MS/MS or fragment spectrum.

In TOF-MS/MS the ions fragment in the field free region of the TOF anddo so either by the process of metastable decay and/or by collision witha neutral gas in a region of high pressure (CID).

When ions fragment in a field free region such as the flight tube orcollision cell, where there are no external forces, the fragmentscontinue with the velocity that is effectively the same as that of theparent (pre-cursor) ion. This in turn means that the energy of thefragment ion is reduced to the fraction of the parent ion energy in theratio of the fragment mass to the parent mass. In other words, thefollowing relationship applies, where E_(f) is the kinetic energy of thefragment ion, E_(p) is the kinetic energy of the parent ion, m_(f) isthe mass of the fragment ion and m_(p) the mass of the parent ion:

E _(f) =E _(p) .m _(f) /m _(p)

With a linear TOF-MS, there is no way of distinguishing between thefragment ions and the parent (pre-cursor) ions because they have thesame velocity and therefore same flight time to the detector. However,as noted above, it is possible to distinguish between fragment ions byusing a reflectron. A reflectron is effectively an energy analyserbecause the distance travelled by the ions into a reflectron isdetermined by the point at which the electrostatic potential is equal tothe kinetic energy of the ions as they enter the reflectron. Forfragment ions the distance travelled into the reflectron is a functionof the energy which is determined by the ratio of the fragment mass tothe parent mass. Since the flight time through the reflectron isdependent on the distance travelled into the reflectron, thetime-of-flight of the fragment ion becomes a function of the ratio ofthe fragment mass to the parent mass.

In principle therefore any reflectron is capable of producing aTOF-MS/MS spectrum. However, because the parent ions have an initialenergy distribution the fragment ions also have an energy distribution.The relationship between the nominal ion energy and the distance fromthe reflectron to the detector at which ions with differing initialenergies are focussed, depends on the shape of the field or voltagedistribution in the reflectron. The most common reflectrons have voltagedistributions which vary linearly from the front to the back. Often (tomake them more compact) there are two or more sections in onereflectron, each with different voltage gradients. For such linear fieldreflectrons the distance between the reflectron and the appropriatelocation of the detector also varies linearly with the nominal ionenergy. It follows that the position of the detector for optimum massresolution will vary linearly with fragment mass. However, in practice,because the detector is a fixed distance from the reflectron, the massresolution for fragments falls rapidly as the mass reduces from theparent mass. The result is that a linear field reflectron cannot on itsown produce a TOF-MS/MS spectrum where the complete fragment mass rangeis in focus and has good mass resolution.

Early instruments got around this problem by stepping the reflectronvoltage and analysing a small segment of the fragment spectrum at atime. The major disadvantage of this was the need to collect multiplespectra and then ‘stitch’ them together which results in long experimenttimes and high sample consumption.

Recently, manufacturers have got around this problem by re-acceleratingthe ions after the point where fragmentation has taken place so that therange of fragment energy is effectively compressed into the narrow rangefor which good mass resolution is produce by the linear reflectron. Socalled TOF/TOF instruments [see for example U.S. Pat. No. 6,512,225(Vestal) and U.S. Pat. No. 6,703,608 (Holle)] either start with, or slowdown the ions to, a low energy typically 1 keV to 8 keV and thenre-accelerate them by means of a second pulsed extraction region to anominal energy around or greater than 20 keV. Such instruments have thedisadvantages of being complex and expensive because of the additionalpulsed high voltage fields required.

An alternative method is to use a reflectron where the potentialdistribution is non-linear so that the range of distance to the detectorfor different fragment ion mass is much smaller than for a linearreflectron. Such a reflectron is known as the curved field reflectron asdescribed in U.S. Pat. No. 5,464,985 (Cotter). In this case it ispossible to measure a complete TOF-MS/MS spectrum with good fragment ionmass resolution without re-acceleration of the fragment ions. Ionstherefore have nominal energies of 20 keV from the source through to thereflectron. This method has the advantages of lower complexity and costbut also allows higher initial energies and therefore higher collisionenergies if using CID. However, one disadvantage is that the best massresolution that can be achieved for the fragment ions is not as high asthose instruments in which re-acceleration is used.

In the case of fragment ions produced by metastable decay (post-sourcedecay, PSD), the production of the fragment ions relies on excessinternal energy in the pre-cursor ions to cause the pre-cursor ions tofragment. The extra energy is produced in a MALDI ion source byincreasing the laser fluence to well above the threshold required forion generation.

In the case of collision induced dissociation (CID) the fragmentation iscaused by high energy collisions with the neutral gas molecules.However, for efficient CID from a MALDI ion source, the laser powerstill has to be above the threshold level.

A consequence of the extra laser power required for TOF-MS/MS is thatthe mass resolution of the pre-cursor ions, and therefore also of thefragment ions, is much lower than for TOF-MS where the laser power isclose to the threshold.

U.S. Pat. No. 5,739,529 (Laukien) describes a method for compensatingthe axial spatial distribution in reflectron TOF-MS. There, a pulsedelectrostatic field is applied using electrodes located either in thereflectron or between the reflectron and the detector to focus thespatial distribution at the detector. This method provides for animprovement in mass resolution for TOF-MS ions over a very narrow massrange.

However, the present inventors have noted that this method is notsuitable to compensate the spatial distribution for TOF-MS/MS becausethe fragment ions are separated in time by the reflectron so that only anarrow mass range of fragments could be focussed.

The present invention seeks to address this and other drawbacksassociated with known methods of performing TOF-MS/MS described above.

SUMMARY OF THE INVENTION

The present inventors have noted that the observed reduction in massresolution for TOF-MS/MS is due to the increase not only in the velocityand radial spatial distributions in the ion source but also the axialspatial distribution. The axial spatial distribution cannot becompensated for by pulsed extraction without losing the focussing of theinitial velocity distribution and it cannot be compensated by the DCelectrostatic fields as used for collimation of the ion beam through theTOF.

In particular, the present inventors have noted that the increased laserpower needed for TOF-MS/MS leads to an increase in axial spatialdistribution.

As is discussed below, the present invention provides a method andapparatus which improve the mass resolution of TOF-MS/MS by compensatingfor the effect of the axial spatial distribution of the ion sourcewithout affecting the other distributions such as the velocitydistribution.

The present invention is particularly concerned with a method andapparatus for focussing the initial axial spatial distribution of ionsin a reflectron time of flight mass spectrometer where the ions havealready been extracted from the ion source using pulsed extraction tocompensate for the initial velocity distribution.

At its most general, the present invention proposes that a pulsedelectrostatic field can be applied to the ions at a point in the fieldfree region where the velocity distribution comes to a spatial focus andthe ions are axially dispersed due only to the initial axial spatialdistribution. As will be clear from the discussion of the presentinvention herein, particular advantages can be achieved in combinationwith a curved-field reflectron such that high mass resolution isachieved for TOF-MS/MS.

This method for improving TOF-MS/MS mass resolution by pulsing anelectrostatic field at the velocity distribution space focus is termedherein as “axial spatial distribution focussing” or “ASDF”.

In a first aspect, the present invention provides a mass spectrometerincluding

-   -   an ion source for generating pre-cursor ions,    -   ion fragmentation means for generating fragment ions from the        pre-cursor ions,    -   a reflectron for focusing the kinetic energy distribution of the        ions, and    -   an ion detector    -   wherein the mass spectrometer also includes    -   axial spatial distribution focusing means which in use acts on        the ions after the ion fragmentation means and before the        reflectron,    -   the axial spatial distribution focusing means being operable to        reduce the spatial distribution of the ions in the direction of        the ion optical axis of the spectrometer.

Preferably the axial spatial distribution focusing means is operable toreduce the axial spatial distribution of the ions such that fragmentions of the same mass arrive at the detector at substantially the sametime as each other.

Preferably the axial spatial distribution focusing means include meansfor generating an axial electrostatic field whereby the electrostaticpotential decreases away from the ion source in an axial direction.

Preferably the axial spatial distribution focusing means include meansfor generating an axial electrostatic field whereby the electrostaticpotential increases away from the ion source in an axial direction.

Preferably the means for generating an axial electrostatic fieldincludes a pair of electrodes spaced from each other in the axialdirection. Suitably the electrodes are separated by a distance of 2 mmto 20 mm, preferably 2 mm to 10 mm, more preferably 2 mm to 5 mm.

Preferably the means for generating an axial electrostatic field isoperable to apply a high voltage pulse to the electrode nearest to theion source whilst maintaining the other electrode at approximately zerovolts potential.

Suitably a voltage in the range 1 kV to 10 kV is applied to theelectrode, more preferably 5 kV to 9 kV. These ranges are particularlypreferred for a spacing between the electrodes of about 5 mm.

Preferably the means for generating an axial electrostatic field isoperable to apply a high voltage pulse to the electrode furthest fromthe ion source whilst maintaining the other electrode at approximatelyzero volts potential.

Preferably the means for generating an axial electrostatic field isoperable to apply the high voltage pulse at a time when the pre-cursorions are at or have just passed the electrode nearest to the ion source.

Preferably the means for generating an axial electrostatic field isoperable to apply the high voltage pulse at a time when the pre-cursorions are between the pair of electrodes.

Preferably the means for generating an axial electrostatic field isoperable to apply the high voltage pulse at a time when the pre-cursorions are at or have just passed the electrode furthest from the ionsource.

Preferably the means for generating an axial electrostatic field isoperable to maintain the high voltage pulse until at least all thepre-cursor and fragment ions have passed through the axial spatialdistribution focusing means.

Suitably the axial electrostatic field (and hence voltage pulse) ismaintained for a period of 5 μs to 50 μs, more preferably 5 μs to 20 μs,and most preferably 10 μs to 15 μs. The duration of the axialelectrostatic field is in practice selected based on the parent ion massto charge ratio and the initial ion energy.

Suitably, the mass spectrometer includes control means to control theaxial electrostatic field. Suitably the control means is a processor orcomputer. Preferably the control means coordinates (e.g. synchronises)the operation of the axial electric field with the generation and/orextraction of ions from the ion source such that the axial electrostaticfield is switched on and off at the appropriate time with respect to theions of interest. Suitably the control means provides (e.g. calculatesand/or retrieves from a memory) the delay between generation and/orextraction of ions from the ion source and operation of the axialelectrostatic field.

Preferably the mass spectrometer includes an electrode located betweenthe axial spatial distribution focusing means and the reflectron, whichelectrode in use acts to terminate the axial electrostatic fieldproduced by the axial spatial distribution focusing means.

Preferably the ion source is a pulsed extraction source which in usefocuses the kinetic energy distribution of the pre-cursor ions so thatfragment ions of the same mass arrive at the detector at substantiallythe same time.

Preferably the axial spatial distribution focusing means are locatedapproximately at the space focus point for the velocity distributionproduced by the ion source.

In practice, there is some tolerance in the respective locations of thespace focus and the point where the ions are when the ASDF pulse isapplied. Suitably the axial spatial distribution focusing means islocated 10 mm or less from the space focus, preferably 5 mm or less,more preferably 3 mm or less and most preferably 1 mm or less.

Pulsed extraction of ions from the ion source can be used to produce aspace focus where all the ions with different velocities in the ionsource are brought to a single point at the same time. At this point,the ions will have an axial spatial distribution that is due only to theaxial spatial distribution in the ion source. By applying a pulsedelectrostatic field at this space focus, the present inventors havefound that the ions acquire an additional velocity distribution thatcorresponds to the initial axial spatial distribution. This arrangementis particularly advantageous because there is no change in the originalvelocity distribution due to the pulsed electrostatic field because theions are at the space focus. The strength of the electrostatic field canbe adjusted so that the extra velocity distribution causes a secondspace focus at the detector. As this is the same position as the spacefocus for the velocity distribution, all the ions within both theinitial velocity distribution and the initial axial spatial distributionarrive at the detector at the same time. As a result, the width of thepeak for one nominal mass to charge is reduced and the mass resolutionimproved accordingly.

Preferably the reflectron is either a curved field reflectron or aquadratic field reflectron.

Where the reflectron is a curved field reflectron it is found that, notonly are the peak widths reduced for the TOF-MS or pre-cursor ions butalso the peak widths of the TOF-MS/MS or fragment ions produced from thefocussed pre-cursor. This is because the fragment ions have the samenominal velocity as the pre-cursor ions and thus the same velocitydistribution and the curved field reflectron is designed so that thespace focus for the fragment ions is close to that of the pre-cursorions.

Whilst curved field reflectrons are preferred, other reflectrons can beused to produce similar behaviour to the curved field reflectron in thatthe space focus at the detector for fragment ions is nominally the sameas or very close to the that of the parent ions. Examples of theseinclude field shapes that are substantially quadratic such as describedby U.S. Pat. No. 4,625,112 (Yoshida) and U.S. Pat. No. 7,075,065(Colburn). It would be possible to achieve results comparable with thecurved-field reflectron by using ASDF with these types of reflectron.Similarly, any other type of reflectron capable of producing nearcoincident focuses for fragment ions and parent ions, could also be usedwith the ASDF method.

Preferably the ion fragmentation means is a collision-induceddissociation (CID) device.

Preferably the spectrometer includes an ion gate for selecting ions of adesired mass such that only ions of the desired mass pass through theion gate, wherein the ion gate is located between the ion source and theaxial spatial distribution focusing means.

Preferably the ion gate is operable in a first mode in which ions areprevented from passing through the ion gate and in a second mode inwhich ions are able to pass through the ion gate. Suitably the ion gateis switched between first and second modes so as to select pre-cursorions of desired mass range. Preferably the switching and selection ofpre-cursor ions is repeated so that multiple sets of precursor ions canbe fragmented and analysed from the same ion pulse.

Thus, the present invention can be used to collect TOF-MS/MS spectrafrom more than one precursor at a time. This has the advantage thatMS/MS data for multiple precursors can be acquired without having torepeat the TOF-MS/MS experiment for each individual precursor. Thisreduces both the total experiment time and sample consumption. As thepre-cursor ions (and their fragment ions with them) pass through theflight tube they separate according to their mass. The precursor ionmass is selected with a pulsed ion gate which is switched off for thetime the precursor ions are within the gate. By switching the ion gateoff multiple times it is possible to transmit multiple precursor ions(and their fragments) in order of mass, lowest first. When the lowestmass precursor ions reach the ASDF pulser it is pulsed on to focus theaxial spatial distribution as appropriate to that precursor. The ASDFpulser is switched off again until the next precursor arrives at whichpoint the pulser is switched on with electrostatic field appropriate tothe new precursor. The TOF-MS/MS spectrum of each precursor is detectedafter being separated and focussed by the curved field reflectron.

In practice the TOF-MS/MS spectra from adjacent precursors may overlapin time. The degree of overlap will depend on the difference in flighttime of the precursors. Where the overlap occurs could cause confusionof the fragments from different precursors. There are several possibleways to reduce the effect of overlap. Firstly, the separation in mass ofthe precursors can be set to a minimum value by selective switching ofthe ion gate so that the overlap between adjacent precursors is limitedto a practical range of the fragment mass. Secondly, it is possible todistinguish low mass fragments of one precursor from the high massfragments of the next through the difference in peak width or massresolution. Thirdly, because the fragment calibration is valid only forfragments of the precursor from which they originated, it is possible todistinguish the correct fragments from the isotope spacing. This will bea particular value, not necessarily 1 Da, only when the fragments havethe calibration for the appropriate precursor.

In a further aspect, the present invention provides a method forperforming mass spectrometry including, in order, the following steps:

-   -   (a) generating pre-cursor ions from an ion source,    -   (b) generating fragment ions from the pre-cursor ions using ion        fragmentation means,    -   (c) reducing the spatial distribution of some or all of the ions        with respect to the axial direction of the spectrometer,    -   (d) focusing the kinetic energy distribution of the ions using a        reflectron,    -   (e) detecting the ions at a detector.

Preferably the axial spatial distribution is reduced such that fragmentions of the same mass arrive at the detector at substantially the sametime as each other.

Preferably the axial spatial distribution is reduced by generating anaxial electrostatic field whereby the electrostatic potential decreasesaway from the ion source in an axial direction.

Preferably the axial spatial distribution is reduced by generating anaxial electrostatic field whereby the electrostatic potential increasesaway from the ion source in an axial direction.

Preferably the axial electrostatic field is provided by a pair ofelectrodes spaced from each other in the axial direction and a highvoltage pulse is applied to the electrode nearest to the ion sourcewhilst maintaining the other electrode at approximately zero voltspotential.

Preferably the axial electrostatic field is provided by a pair ofelectrodes spaced from each other in the axial direction and a highvoltage pulse is applied to the electrode furthest from the ion sourcewhilst maintaining the other electrode at approximately zero voltspotential.

Preferably the high voltage pulse is applied at a time when thepre-cursor ions are at or have just passed the electrode nearest to theion source.

Preferably the high voltage pulse is applied at a time when thepre-cursor ions are between the pair of electrodes.

Preferably the high voltage pulse is applied at a time when thepre-cursor ions are at or have just passed the electrode furthest fromthe ion source.

Preferably the high voltage pulse is maintained until at least all thepre-cursor and fragment ions have passed through the pair of electrodes.

Preferably the ion source is a pulsed extraction source which focusesthe kinetic energy distribution of the pre-cursor ions so that fragmentions of the same mass arrive at the detector at substantially the sametime.

Preferably the step of reducing the spatial distribution of some or allof the ions with respect to the axial direction of the spectrometeroccurs at the space focus point for the velocity distribution producedby the ion source.

Preferably the method includes selecting ions of a desired mass rangeprior to reducing the spatial distribution in the axial direction.

Preferably the ions of desired mass range are selected by providing anion selecting electrostatic field to prevent ions from passing along thespectrometer in an axial direction the detector and switching off theion selecting electrostatic field to allow ions of the desired massrange to pass along the spectrometer in the axial direction.

Preferably the method includes the steps of (i) selecting a first set ofions having a first desired mass range and reducing the spatialdistribution of the first set of ions in the axial direction of thespectrometer, and (ii) selecting a second set of ions having a seconddesired mass range and reducing the spatial distribution of the secondset of ions in the axial direction of the spectrometer.

All of the optional and/or preferred features of any one aspect of thisinvention may be applied to any one of the other aspects. In particular,the optional and preferred features associated with the spectrometeraspect also apply to the method aspect, and vice versa. Any one aspectof this invention may be combined with any one or more of the otheraspects.

Embodiments and experiments relating to the present invention arediscussed below, with reference to the accompanying drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the radial spatial distribution in the ion source;

FIG. 2 shows the angular distribution in the ion source;

FIG. 3 shows the axial velocity distribution in the ion source;

FIG. 4 shows the axial spatial distribution in the ion source;

FIG. 5 shows a schematic of an ASDF pulser just before the pre-cursorand fragment ions enter;

FIG. 6 shows a schematic of ASDF pulser just after the pre-cursor andfragment ions enter;

FIG. 7 shows a block schematic of an embodiment of the presentinvention;

FIG. 8 shows initial ion trajectories for an ion model;

FIG. 9 shows ion trajectories through an ASDF pulser towards a curvedfield reflectron and back to a detector;

FIG. 10 shows the peak width and mass resolution of fragment ions ofpre-cursor ACTH 18-39 (m/z 2466 Da) for 50 μm axial spatial distributionwithout ASDF;

FIG. 11 shows peak width and mass resolution of fragment ions ofpre-cursor ACTH 18-39 (m/z 2466 Da) for 50 μm axial spatial distributionwith ASDF; and

FIG. 12 shows a comparison of mass resolution for fragments ofpre-cursor ACTH 18-39 (m/z 2466 Da) for an axial spatial distribution of50 μm, with and without ASDF.

In the embodiment shown in FIG. 5, the ASDF pulser 50 consists of a cellwith two electrodes 52, 54 which may be apertures or high transmissiongrids. In this embodiment, the electrodes are spaced apart by a few mm,but other spacings are possible, for example 2 mm to 20 mm. Although notshown in FIG. 5, the pulser 50 is positioned at a point in the flighttube that is after the CID cell and after the point at which formationof fragment ions by meta-stable decay occurs but before the reflectron.As is preferred, the pulsed extraction at the ion source is arranged sothat the space focus point for the initial velocity distribution is ator close to the position of the ASDF pulser. A pulsed electrostaticfield is generated by applying a high voltage pulse 60 to the firstelectrode 52 at the time when the pre-cursor ions of interest 56, 58have just passed into the pulser 50. The second electrode 54 ismaintained at 0V during this time.

An appropriate electrostatic field sufficient to focus the initial axialspatial distribution is produced by adjusting the amplitude of thevoltage pulse on the first electrode 52. Thus, as shown in FIG. 6, apotential V₁ is applied to the first electrode 52 when all of the ionsof interest have passed into the cell (i.e. moved past the firstelectrode). Suitable voltages are in the range 1 kV to 10 kV, morepreferably 5 kV to 9 kV. The voltage is maintained until at least thetime when all the pre-cursor and fragment ions have passed through thepulser. Suitable pulse durations are 5 μs to 50 μs, 5 μs to 20 μs, and10 μs to 15 μs. During this time, the second electrode 54 is maintainedat 0V, so that the electrostatic potential within the pulser variesalong the ion optical axis (i.e. in the axially direction). Thus, anaxial electrostatic field is provided during the time that the ions ofinterest are within the pulser. As can be appreciated from FIG. 6, theelectrostatic potential is higher closer to the first electrode andlower at increasing distances from the first electrode. This potentialgradient between the electrodes means that ions closer to the firstelectrode will experience an acceleration for longer than ions closer tothe second electrode. In this way, ions of interest arriving at thepulser later will experience a greater increase in velocity than thosethat arrived earlier. This causes the ions of interest to bunchtogether, thereby reducing or eliminating the initial axialdistribution.

In this and other embodiments, the polarity of the applied potential canbe positive or negative, so that the axial electrostatic fieldaccelerates or decelerates the ions.

In a further embodiment, the arrangement is identical to that shown inFIGS. 5 and 6 except that the high voltage pulse is applied to thesecond electrode 54 whilst the first electrode is grounded. Furthermore,in one mode of operation, the timing of the pulse is such that the pulseis applied when the ions of interest are behind the second electrode 54(e.g. located between the first and second electrodes). In a furthermode of operation, the pulse is applied when the ions of interest are infront of the second electrode 54 (i.e. between the second electrode andthe detector).

In the further embodiment wherein the pulse is applied to the secondelectrode, there is also a third, grounded, electrode located after thesecond electrode so that the axial electrostatic field is terminatedproperly.

A block diagram of the complete TOF-MS/MS instrument 70 is shown in FIG.7. The ASDF pulser/cell 72 is located between the CID cell 74 and thereflectron 76. Thus, pre-cursor ions generated from MALDI source 78 havean initial axial spatial distribution, pass through linear TOF 80 andexperience collision induced dissociation in CID cell 74 to producefragment ions (which possess the initial axial spatial distribution ofthe pre-cursor ions). The fragment ions then pass through ASDFpulser/cell 72 where an axial electrostatic field is applied to the ionsto impart a corrective velocity to the ions such that the ions arefocused (that is, no longer have the initial axial spatial distribution)at the entrance to reflectron 82.

The effectiveness of the invention can be illustrated with iontrajectory modelling of a time of flight mass spectrometer (SIMION 3 dV8). FIG. 8 shows the initial trajectories of parent (pre-cursor) ionsfrom three points 90 on a sample surface corresponding to a radialspatial distribution of 100 μm, angular distribution of 30° and an axialvelocity distribution of 350 to 650 ms⁻¹. Also included are ions 92 withthe same initial trajectories but starting at a point 50 μm above thesample surface to represent the axial spatial distribution caused byincreasing the laser power to produce MS/MS ions (and/or to representions generated from thick samples).

FIG. 9 shows the trajectory of the fragment ions with 50% of the parentmass at the detector 100 following pulsed extraction of the parent ions,CID to form fragment ions, ASDF pulsing (in ASDF cell 102) and thecurved-field reflectron (not shown).

The graph of FIG. 10 shows the peak width at the detector and thecorresponding mass resolution for the different mass fragments of thepeptide ACTH 18-39 with a nominal mass to charge ratio of 2466 Da butwith the mass spectrometer set up for best mass resolution without usingthe ASDF pulser. It can be seen that the peak widths are typicallyaround 14 ns corresponding to mass resolution for the fragment ionswhich is less than 2000. This mass resolution would not be good enoughto resolve the isotope distribution of the fragment ions.

The graph of FIG. 11 shows the results for the same ions but in thiscase with the ion source pulsed extraction tuned to produce a spacefocus in the ASDF pulser and the ASDF pulser with 9 kV pulses applied tothe first electrode (a grid, but the electrode could have another form,for example an aperture). The 9 kV pulse is applied to the firstelectrode after the fragment ions have entered the pulser. In this casethe peak widths have been reduced to around 2 ns with fragment massresolution up to a maximum of 10,000. This resolution corresponds topeak width for the fragments of about 0.25 Da which is enough to easilyseparate individual peaks in the fragment isotope distributions.

A direct comparison of the TOF-MS/MS mass resolution for this example,with and without ASDF, is shown in FIG. 12. Clearly, a considerableimprovement in the mass resolution is achieved for the entire range offragment masses.

1-18. (canceled)
 19. A mass spectrometer including an ion source forgenerating pre-cursor ions, ion fragmentation means for generatingfragment ions from the pre-cursor ions, a reflectron for focusing thekinetic energy distribution of the fragment ions, and an ion detectorwherein the mass spectrometer also includes axial spatial distributionfocusing means which in use acts on the ions after the ion fragmentationmeans and before the reflectron, the axial spatial distribution focusingmeans being operable to reduce the spatial distribution of the ions inthe direction of the ion optical axis of the spectrometer.
 20. A massspectrometer according to claim 19 wherein the axial spatialdistribution focusing means is operable to reduce the axial spatialdistribution of the ions such that fragment ions of the same mass arriveat the detector at substantially the same time as each other.
 21. A massspectrometer according to claim 19 wherein the axial spatialdistribution focusing means include means for generating an axialelectrostatic field whereby the electrostatic potential decreases awayfrom the ion source in an axial direction or increases away from the ionsource in an axial direction.
 22. A mass spectrometer according to claim21, wherein the means for generating an axial electrostatic fieldincludes a pair of electrodes spaced from each other in the axialdirection.
 23. A mass spectrometer according to claim 22 wherein themeans for generating an axial electrostatic field is operable to apply ahigh voltage pulse to the electrode nearest to the ion source whilstmaintaining the other electrode at approximately zero volts potential.24. A mass spectrometer according to claim 22 wherein the means forgenerating an axial electrostatic field is operable to apply a highvoltage pulse to the electrode furthest from the ion source whilstmaintaining the other electrode at approximately zero volts potential.25. A mass spectrometer according to claim 23, wherein the means forgenerating an axial electrostatic field is operable to apply the highvoltage pulse at a time when the pre-cursor ions are at or have justpassed the electrode nearest to the ion source.
 26. A mass spectrometeraccording to claim 24, wherein the means for generating an axialelectrostatic field is operable to apply the high voltage pulse at atime when the pre-cursor ions are between the pair of electrodes.
 27. Amass spectrometer according to claim 24, wherein the means forgenerating an axial electrostatic field is operable to apply the highvoltage pulse at a time when the pre-cursor ions are at or have justpassed the electrode furthest from the ion source.
 28. A massspectrometer according to claim 23, wherein the means for generating anaxial electrostatic field is operable to maintain the high voltage pulseuntil at least all the pre-cursor and fragment ions have passed throughthe axial spatial distribution focusing means.
 29. A mass spectrometeraccording to claim 19, wherein the mass spectrometer includes anelectrode located between the axial spatial distribution focusing meansand the reflectron, which electrode in use acts to terminate the axialelectrostatic field produced by the axial spatial distribution focusingmeans.
 30. A mass spectrometer according claim 19 wherein the ion sourceis a pulsed extraction source which in use focuses the kinetic energydistribution of the pre-cursor ions so that fragment ions of the samemass arrive at the detector at substantially the same time.
 31. A massspectrometer according to claim 19 wherein the axial spatialdistribution focusing means are located approximately at the space focuspoint for the velocity distribution produced by the ion source.
 32. Amass spectrometer according to claim 19 wherein the reflectron is eithera curved field reflectron or a quadratic field reflectron.
 33. A massspectrometer according to claim 19 wherein the ion fragmentation meansis a collision-induced dissociation (CID) device.
 34. A massspectrometer according to claim 19 wherein the spectrometer includes anion gate for selecting ions of a desired mass such that only ions of thedesired mass pass through the ion gate, wherein the ion gate is locatedbetween the ion source and the axial spatial distribution focusingmeans.
 35. A mass spectrometer according to claim 34, wherein the iongate is operable in a first mode in which ions are prevented frompassing through the ion gate and in a second mode in which ions are ableto pass through the ion gate.
 36. A method for performing massspectrometry including, in order, the following steps: (a) generatingpre-cursor ions from an ion source, (b) generating fragment ions fromthe pre-cursor ions using ion fragmentation means, (c) reducing thespatial distribution of some or all of the ions with respect to theaxial direction of the spectrometer, (d) focusing the kinetic energydistribution of the fragment ions using a reflectron, (e) detecting theions at a detector.
 37. A method according to claim 36 wherein the axialspatial distribution is reduced such that fragment ions of the same massarrive at the detector at substantially the same time as each other. 38.A method according to claim 36 wherein the axial spatial distribution isreduced by generating an axial electrostatic field whereby theelectrostatic potential decreases away from the ion source in an axialdirection or increases away from the ion source in an axial direction.39. A method according to claim 38 wherein the axial electrostatic fieldis provided by a pair of electrodes spaced from each other in the axialdirection and a high voltage pulse is applied to the electrode nearestto the ion source whilst maintaining the other electrode atapproximately zero volts potential.
 40. A method according to claim 38wherein the axial electrostatic field is provided by a pair ofelectrodes spaced from each other in the axial direction and a highvoltage pulse is applied to the electrode furthest from the ion sourcewhilst maintaining the other electrode at approximately zero voltspotential.
 41. A method according to claim 39 wherein the high voltagepulse is applied at a time when the pre-cursor ions are at or have justpassed the electrode nearest to the ion source.
 42. A method accordingto claim 40, wherein the high voltage pulse is applied at a time whenthe pre-cursor ions are between the pair of electrodes.
 43. A methodaccording to claim 40, wherein the high voltage pulse is applied at atime when the pre-cursor ions are at or have just passed the electrodefurthest from the ion source.
 44. A method according to claim 39,wherein the high voltage pulse is maintained until at least all thepre-cursor and fragment ions have passed through the pair of electrodes.45. A method according to claim 36 wherein the ion source is a pulsedextraction source which focuses the kinetic energy distribution of thepre-cursor ions so that fragment ions of the same mass arrive at thedetector at substantially the same time.
 46. A method according to claim36 wherein the step of reducing the spatial distribution of some or allof the ions with respect to the axial direction of the spectrometeroccurs at the space focus point for the velocity distribution producedby the ion source.
 47. A method according to claim 36 wherein the methodincludes selecting ions of a desired mass range prior to reducing thespatial distribution in the axial direction.
 48. A method according toclaim 47 wherein the ions of desired mass range are selected byproviding an ion selecting electrostatic field to prevent ions frompassing along the spectrometer in an axial direction the detector andswitching off the ion selecting electrostatic field to allow ions of thedesired mass range to pass along the spectrometer in the axialdirection.
 49. A method according to claim 47 wherein the methodincludes the steps of (i) selecting a first set of ions having a firstdesired mass range and reducing the spatial distribution of the firstset of ions in the axial direction of the spectrometer, and (ii)selecting a second set of ions having a second desired mass range andreducing the spatial distribution of the second set of ions in the axialdirection of the spectrometer.